Effects of Umbelliferone in a Murine Model of Allergic Airway Inflammation

European Journal of Pharmacology 609 (2009) 126–131

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European Journal of Pharmacology

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Pulmonary, Gastrointestinal and Urogenital Pharmacology Effects of umbelliferone in a murine model of allergic airway inﬂammation

Juliana F. Vasconcelos a,e, Mauro M. Teixeira b, José M. Barbosa-Filho c, Maria F. Agra c, Xirley P. Nunes d, Ana Maria Giulietti e, Ricardo Ribeiro-dos-Santos a,f, Milena B.P. Soares a,f,⁎ a Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, 40296-750, Salvador, BA, Brazil b Instituto de Ciências Biomédicas, Universidade Federal de Minas Gerais, 31270-901, Belo Horizonte, MG, Brazil c Laboratório de Tecnologia Farmacêutica, Universidade Federal da Paraíba, 58051-970, João Pessoa, PB, Brazil d Colegiado do Curso de Medicina, Universidade Federal do Vale do São Francisco, 56306-410, Petrolina, PE, Brazil e Departamento de Ciências Biológicas, Universidade Estadual de Feira de Santana, 44031-640, Feira de Santana, BA, Brazil f Hospital São Rafael. Av. São Rafael, 2152. São Marcos 41253-190, Salvador, BA, Brazil article info abstract

Article history: The therapeutic effects of umbelliferone (30, 60 and 90 mg/kg), a coumarin isolated from Typha domingensis Received 7 December 2008 (Typhaceae) were investigated in a mouse model of bronchial asthma. BALB/c mice were immunized and Received in revised form 19 February 2009 challenged by nasal administration of ovalbumin. Treatment with umbelliferone (60 and 90 mg/kg) caused a Accepted 3 March 2009 marked reduction of cellularity and eosinophil numbers in bronchoalveolar lavage fluids from asthmatic mice. In Available online 14 March 2009 addition, a decrease in mucus production and lung inflammation were observed in mice treated with umbelliferone. A reduction of IL-4, IL-5, and IL-13, but not of IFN-γ, was found in bronchoalveolar lavage fluids of mice treated with Keywords: fi Umbelliferone umbelliferone, similar to that observed with dexamethasone. The levels of ovalbumin-speci cIgEwerenot Coumarin significantly altered after treatment with umbelliferone. In conclusion, our results demonstrate that umbelliferone Typha domingensis attenuates the alteration characteristics of allergic airway inflammation. The investigation of the mechanisms of Bronquic asthma action of this molecule may contribute for the development of new drugs for the treatment of asthma. (Mouse) © 2009 Elsevier B.V. All rights reserved.

1. Introduction Apiaceae (Umbelliferae) family such as carrot, coriander and garden angelica, as well as plants from other families such as the mouse-ear Coumarins constitute a very large class of compounds present in hawkweed. It is a yellowish-white crystalline solid which has a slight several species belonging to different botanical families, which are solubility in hot water, but high solubility in ethanol (Dean, 1963). widespread in the world. They are secondary metabolites naturally Asthma is a chronic inflammatory disorder of the airways in which occurring in different parts of the plants, such as roots, flowers and fruits many cells and elements play a role, associated with increased airway (Leal et al., 2000; Ribeiro and Kaplan, 2002; Razavi et al., 2008). The search hyperresponsiveness, leading to recurrent episodes of wheezing, breath- for useful pharmaceutical has led to a resurgence of interest in coumarins lessness, chest tightness, and coughing (Barnes, 2001). This chronic because these substances display potent and relevant pharmacological inflammation is characterized by infiltration of eosinophils, neutrophils, activities that are structure-dependent, while at the same time appearing mast cells and T cells (Casale et al., 1987; Wenzel, 2003), and is promoted to lack toxicity in mammalian systems (Hoult and Payá, 1996). by Th2 type immune responses (Barnes, 2001; El Biaze et al., 2003; Reed, Among the biological effects of coumarins are anti-microbian (Thati 2006). In the current study we aimed to evaluate the effects of et al., 2007), anti-viral (Mazzei et al., 2008), anti-thrombotic, vasodilatory umbelliferone, a coumarin purified from Typha domingensis (Typhaceae), (Casley-Smith et al., 1993), antitumoral, antineoplasic, antiinflammatory in a mouse model of asthma, as a potential anti-asthmatic agent. (Hoult and Payá, 1996; Lino et al., 1997), and antimetastatic properties (Jiménez-Orozco et al., 2001; Finn et al., 2004; Finn et al., 2005; Elinos- Báez et al., 2005), and inhibition of acetylcholinesterase and angiotensin 2. Materials and methods converting enzyme (Barbosa-Filho et al., 2006a,b). In humans, coumarins have a short half-life, and its major biotransformation product (75%) is 2.1. Plant material umbelliferone (Hoult and Payá, 1996; Egan et al., 1997). Umbelliferone or 7-hydroxycoumarin is a widespread natural product The aerial parts of T. domingensis Pers. were collected for first of the coumarin family (Fig. 1). It occurs in many familiar plants from the extracts in March, 2002 in Bravo, State of Bahia, in temporary shallow lake in Dryland “Caatinga” Bioma and after in December, 2005 near the city of Santa Rita, State of Paraíba, Brazil. The plant was identified ⁎ Corresponding author. Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, by Ana Maria Giulietti and Maria de Fátima Agra and the voucher Laboratório de Engenharia Tecidual e Imunofarmacologia. Rua Waldemar Falcão, 121, Candeal. 40296-710, Salvador, Bahia, Brazil. Tel.: +55 71 3176 2260; fax: +55 71 31762272. specimens (Giulietti 2035 et al. and Agra 5520 et Góis — JPB) were E-mail address: milena@bahia.fiocruz.br (M.B.P. Soares). deposited in the Herbaria HUEFS and JPB.

2.4. Sensitization and challenge with ovalbumin and treatment

Allergic airway inflammation was induced as described before (Vasconcelos et al., 2008). Groups of seven mice received systemic immunization by subcutaneous injection of 10 μg of chicken egg ovalbumin (Grade V, N98% pure; Sigma, St Louis, MO) diluted in Fig. 1. Chemical structure of umbelliferone. 2 mg/ml alum (AlumImject; Pierce, Rockford, IL) followed by a booster injection at day 14. A nasal challenge was performed starting at day 28, by inhalational exposure to aerosolised ovalbumin for 15 min/day, on five consecutive days. Exposures were carried out in an acrylic box. A 2.2. Extraction and isolation of umbelliferone solution of 1% ovalbumin in saline was aerosolised by delivery of compressed air to a sidestream jet nebuliser (RespiraMax, NS, Brazil). The aerial parts (2000 g) of T. domingensis were extracted with 95% Two hours before each aerosol delivery, mice were treated orally with ethanol at room temperature. The extract (150 g) was evaporated under umbelliferone (30, 60, and 90 mg/kg), dexamethasone (30 mg/kg) or vacuum to yield a brown residue which was suspended in methanol: vehicle (10% DMSO in saline). H2O (3:7 v/v) and fractionated with chloroform and ethyl acetate. The chloroform phase (25 g) was subjected to column chromatography over 2.5. Collection of blood and bronchoalveolar lavage silica gel, eluted with hexane, chloroform and methanol mixtures in an increasing order of polarity, yielding three coumarins and one cinnamic Twenty four hours after the last inhalational exposure, mice were acid derivative. The ethyl acetate phase (25 g) was subjected to anesthetized and bled via the brachial plexus for collection of blood chromatography column over Sephadex LH-20 using methanol yielding samples used to estimate the IgE production. Bronchoalveolar lavage fl fi two avonoids. The isolated compounds were identi ed as coumarin (BAL) was performed twice by intratracheal instillation of 1 ml of PBS. (0.0044%), scopoletin (0.0014%) (Brateoeff and Perez-Amador, 1994), The first lavage fluid was centrifuged, and aliquots of the supernatant umbelliferone (0.007%) (Cussans and Huckerby, 1975), p-coumaric acid were kept at 70 °C until use for cytokine measurements. The second (0.0016%), (Pouchert, 1992), quercetin (0.0028%) (Williams et al., 1971) lavage fluid was centrifuged and the two cell pellets were resus- and isorhamnetin-3-O-glucoside (0.0039%) (Bilia et al., 1994) based on pended in a PBS final volume of 1 ml. The number of total leukocytes 1 13 HNMR, C NMR, COSY, HMQC and HMBC spectroscopic data and in bronchoalveolar lavage fluid was estimated in a Neubauer chamber. comparison with those reported in the literature. Differential counts were obtained using panotic-stained cytospin preparations. A differential count of 200 cells was made in a blinded 2.3. Animals fashion and according to standard morphologic criteria.

Male BALB/c mice, 4–6 weeks old, were used in the experiments. 2.6. Histopathological and morphometric analysis All animals were raised and maintained at the animal facilities of the Gonçalo Moniz Research Center, FIOCRUZ/BA, in rooms with con- The right lobe of the lungs from each animal was removed for trolled temperature (22±2 °C) and humidity (55±10%) and histological analysis. The lung was inflated via the tracheal cannula continuous air renovation. Animals were housed in a 12 h light/12 h with 4% buffered formalin, fixed in the same solution, and embedded dark cycle (6 am–6 pm) and provided with rodent diet and water ad in paraffin. Sections were stained with hematoxylin and eosin for libitum. Animals were handled according to the NIH guidelines for quantification of inflammatory cells by optical microscopy. For each animal experimentation. All procedures described here had prior lung 10 fields (400×) were analyzed per section, and the data used to approval from the local animal ethics committee. calculate the mean number of cells per mm2. Mucus production was

Fig. 2. Total cell counts and leukocyte quantification in BAL samples obtained from mice submitted to different treatments. Mice were sacrificed 24 h after the last challenge with ovalbumin. The cellularity in BAL fluid from control or ovalbumin-challenged mice treated with saline, dexamethasone (dexa) or umbelliferone (umb) was evaluated. A, Total cell counts. B, Number of neutrophils in 200 cells. C, Number of eosinophils in 200 cells. D, Number of mononuclear cells in 200 cells. Values are expressed as means±S.E.M. of 6–7 mice per group, in one of two experiments performed. ⁎⁎⁎Pb0.001. 128 J.F. Vasconcelos et al. / European Journal of Pharmacology 609 (2009) 126–131 analyzed in alcian blue-stained sections. All images were digitalized (IFN)-γ in bronchoalveolar lavage fluid were also determined by ELISA using a color digital video camera (CoolSnap cf) adapted to a BX41 using specific antibody kits (R&D System, Minnesota, MN, USA), microscope (Olympus, Tokyo, Japan) calibrated with a reference according to manufacturer's instructions. measurement slide and were analyzed using Image Pro image program (version 6.1; Media Cybernetics, San Diego, CA, USA). 2.8. Statistical analysis

2.7. Ovalbumin-specific IgE antibody levels and cytokine production Results were expressed as means±S.E.M. of 7 mice per group. Statistical comparisons between groups were performed by analysis of IgE antibody levels to ovalbumin in sera samples from individual variance (ANOVA) followed by Newman–Keuls multiple comparison animals were quantified using an enzyme immunoassay modified from test, using GraphPad InStat program (Software Inc., San Diego, CA, USA). Jungsuwadee et al. (2004).Briefly, microplate wells coated with 2 µg/ml Results were considered to be statistically significant when Pb0.05. of anti-mouse IgE (Pharmingem, San Diego, CA) were incubated overnight with dilutions of the test samples in PBS containing 5% non- 3. Results fat milk. Detection of bound anti-ovalbumin IgE was performed by sequential ovalbumin incubation followed by biotinylated anti-ovalbu- 3.1. Treatment with umbelliferone reduces lung inflammation min antibody (Fitzgerald, Concord, MA, USA). After incubation with streptoavidin-peroxidase conjugate, the reaction was developed using Mice sensitized and challenged with ovalbumin have increased the 3,3′,5,5′-tetramethylbenzidine (TMB) peroxidase substrate and read at number of inflammatory cells in bronchoalveolar lavage fluid, compared 450 nm. Concentrations of interleukin (IL)-4, IL-5, IL-13, and interferon to control mice — sensitized and challenged with saline (Fig. 2A). The

Fig. 3. Histopathology of lungs from control and asthmatic mice. Lung sections of control (A–C) and asthmatic mice treated with vehicle (D–F), dexamethasone (G–I), or umbelliferone 90 mg/ml (J–L). Narrow arrows indicate areas of alcian blue+ cells; large arrows indicate inﬂammatory cells. A, D, G, and J: Alcian blue staining. B, C, E, F, H, I, K and L: H&E staining. J.F. Vasconcelos et al. / European Journal of Pharmacology 609 (2009) 126–131 129

Fig. 6. Ovalbumin-speciﬁc IgE antibodies in asthmatic mice. The levels of anti-OVA IgE antibodies were determined in the sera of individual mice by ELISA. The data are expressed as means±S.E.M. of 7 mice per group, in one of two experiments performed. ⁎⁎Pb0.01 compared to saline-treated mice.

potent reduction in the number of eosinophils (Fig. 2C). The number of mononuclear cells was signiﬁcantly altered only by treatment with umbeliferone at 60 mg/ml and by dexamethasone (Fig. 2D).

Fig. 4. Quantification of inflammation and mucus production in lungs of mice. A, Intensity 3.2. Histological evaluation of lungs from umbelliferone-treated mice of inflammation in lungs of mice treated with saline, dexamethasone (dexa) or umbelliferone 60 mg/ml (umb) compared to control animals. The number of To characterize further the changes in lung pathology caused by fl in ammatory cells was evaluated on H&E-stained sections. B, Analysis of mucus antigen challenge of immunized mice, we examined lung sections stained production on alcian blue-stained lung sections. The area of alcian blue staining was fi estimated by morphometric analysis. Data are expressed as means±S.E.M. of 7 mice per with H&E. Ovalbumin challenge caused an intense cell in ltrate containing group, in one of two experiments performed. ⁎⁎⁎Pb0.001 compared to saline-treated mice. many lymphocytes, macrophages, and eosinophils (Fig. 3EandF).Mice treated with umbelliferone at 60 and 90 mg/ml, but not at 30 mg/ml, had reduced inflammation, particularly reduced eosinophil infiltration, effects of umbelliferone treatment in lung inflammation were evaluated although some less dense inflammatory foci remained (Fig. 3KandL; comparing bronchoalveolar lavage fluid cytology of umbelliferone- Fig. 4A). Dexamethasone treatment almost completely eliminated the treated mice to that of mice treated with vehicle. The number of total inflammatory infiltrate, in particular eosinophils (Fig. 3HandI;Fig. 4A). cells and of neutrophils in the bronchoalveolar lavage fluid was Lung sections were stained with alcian blue to analyze mucus significantly reduced after umbelliferone treatment compared to vehi- overproduction. Lungs from control mice revealed rare alcian blue+ cle-treated mice (Fig. 2AandB).Asignificant inhibition was observed in cells in the respiratory epithelium compared to saline-treated group, mice treated with dexamethasone, a gold standard drug. Treatment with which had high alcian blue+ staining (Fig. 3AandD;Fig. 4B). Similar umbelliferone at 60 and 90 mg/ml, but not at 30 mg/ml, decreased to dexamethasone, umbelliferone treatment at 60 mg/ml was significantly the number of eosinophils found in the bronchoalveolar capable of modulating mucus production, decreasing the intensity lavage fluid of challenged mice, although dexamethasone caused a more of alcian blue+ staining observed in the saline-treated group by 38%

Fig. 5. Cytokine production in umbelliferone-treated mice. Cytokine levels in individual mice from each experimental group were determined in BAL samples by ELISA. A, IL-4. B, IL-5. C, IL-13. D, IFN-γ. Data are expressed as means±S.E.M. of 6–7 mice per group. ⁎⁎PbB0.01; ⁎⁎⁎Pb0.001 compared to saline-treated mice. 130 J.F. Vasconcelos et al. / European Journal of Pharmacology 609 (2009) 126–131 and a more potent effect after umbelliferone treatment at 90 mg/ml The effects of umbelliferone in the reduction of histological (Fig. 3GandJ;Fig. 4B). alterations and cellularity in the bronchoalveolar lavage fluid were similar to those of dexamethasone, a synthetic glucocorticoid 3.3. Treatment with umbelliferone modulated T-helper type 2 cytokine commonly used as a gold standard anti-inflammatory drug. The side response but not IgE production effects after chronic use of corticoids, such as osteopenia, poor wound healing, hyperglycemia, hypertension, and cataracts limit their The production of cytokines was studied in bronchoalveolar lavage of systemic administration (Barnes, 2001; Schimmer & Parker, 2001), individual mice from each group. As expected, the concentrations of IL-4, and therefore the search of substances with similar immunosuppres- IL-5, and IL-13 were increased in ovalbumin-immunized, compared to sive activities, free or with lower toxicity, is of great interest (Marinho control mice (Fig. 5A–C). Treatment with umbelliferone caused a et al., 2004; Bezerra-Santos et al., 2006; Costa et al., 2008a,b). reduction in the levels of Th2-associated cytokines, compared to saline- Although reports of adverse effects in humans resulting from treated group, an effect similar to that of dexamethasone. The levels of coumarin administration are rare (Casley-Smith et al., 1993; Lake, Th1-associated cytokine IFN-γ were not increased by ovalbumin 1999), it is acutely toxic to laboratory animals in chronic oral gavage challenge, but were reduced by dexamethasone. In contrast, umbellifer- administration at doses equal or higher of 200 mg/kg (Born et al., one did not alter the production of IFN-γ (Fig. 5D). 2003). There are marked differences in coumarin metabolism When levels of ovalbumin-specific IgE antibodies were analyzed, between susceptible rodent and other species, including humans. ova-immunized mice treated with vehicle had high levels of serum According to Lake (1999) the 7-hydroxylation pathway of coumarin anti-ovalbumin IgE antibodies compared to control mice (Fig. 6). A metabolism, which generates umbelliferone, is usually the major significant reduction in ovalbumin-specific IgE antibodies was pathway in humans but not in rats and mice, in which it represents observed in mice treated with dexamethasone, but not with only a minor pathway of metabolism. In contrast, the major route of umbelliferone, compared to saline-treated controls (Fig. 6). coumarin metabolism in murines is by a 3,4-epoxidation pathway, which results in the formation of toxic metabolites. In our study we 4. Discussion did not observe any signs of toxicity in umbelliferone-treated mice. Carrageenan stimulates the release of several inflammatory mediators In the present study we demonstrated a potent anti-inflammatory such as histamine, serotonin, bradykinin and prostaglandins. Non- activity of umbelliferone in an allergic airway inflammation model steroidal anti-inflammatory drugs (NSAID) block the synthesis of induced by ovalbumin administration in mice. This effect was evidenced prostaglandins by inhibiting cyclooxygenase (COX) (Hoult et al., 1994). by a marked reduction of inflammation both in bronchoalveolar lavage Murakami et al. (2000) demonstrated umbelliferone does not suppress − fluid and in the lung, especially in neutrophil and eosinophil numbers, in O2 generation in vitro nor attenuates lypopolysaccharide-induced nitric mucus production and in the levels of Th2-associated cytokines in oxide synthase (NOS)/COX-2 expression and TNF-α release. Interesting, bronchoalveolar lavage after umbelliferone treatment. umbelliferone inhibits prostaglandin biosynthesis, which involves fatty Umbelliferone is one of several coumarins with anti-inflammatory acid hydroperoxy intermediates, indicating that this substance has a activity already reported (Hoult and Payá 1996; Lino et al., 1997). In our mechanism of action similar to NSAID in a carrageenan-induced study it was extracted from T. domingensis (Typhaceae), a species widely inflammation model (Fylaktakidou et al., 2004). This is an unlikely distributed in north-east Brazil, especially found in damp areas and in mechanism of action of umbelliferone in our system, NSAID are not shown shallow water, lakes and river margins, used for water and soil metal to inhibit effectively the migration of eosinophils and mucus production in decontamination (Taggart et al., 2005; França, 2006; Maine et al., 2007). murine models of allergic inflammation. We have isolated three coumarins from T. dominguensis. The most There are certain pharmacologic compounds that inhibit eosino- abundant was identified as coumarin or 1,2-benzopyrone with effects phil function, eosinophil infiltration, or both, which is desirable for the already described in an experimental asthma model (Marinho et al., treatment of patients with atopy/ allergic disorders (Barnes, 2001). 2004; Biavatti et al., 2004; Zhang et al., 2005; Dos Santos et al., 2006; The inhibition of Type-II cytokine production by umbelliferone Werz, 2007). The other two were scopoletin, isolated in small amounts, suggests an inhibition of T cells and may be underlining the effects and umbelliferone, which has not been studied in animal models of of the drug on the system, as allergic airway inflammation is shown to asthma before. depend on the action of IL-4, IL-5 and IL-13 (Bodey et al., 1999; Boyton Umbelliferone is present in Typhae Pollen, a traditional Chinese herbal and Altmann, 2004). It has also been shown that IL-5 enables terminal medicine with demonstrated immunomodulatory activity (Park et al., differentiation and proliferation of eosinophil precursors, as well as 2004; Qin & Sun, 2005). Thus, umbelliferone may be one of the substances the eosinophil activation effect (Barnes, 2001). The reduction of responsible for the anti-inflammatoryeffectsofthisextract.Arecent eosinophil infiltrates found in umbelliferone-treated asthmatic mice study of some plant species largely used in north-eastern Brazil for may be due to IL-5 reduction, since it promotes the differentiation to respiratory tract diseases has shown they have in common coumarins as and activation of eosinophils. Further studies are clearly needed to one of their active principles (Leal et al., 2000; Agra et al., 2007). understand further the ability of umbelliferone to inhibit the synthesis Ramanitrahasimbola et al. (2005) showed that the bronchodilator effect of these cytokines. after treatment with Phymatodes scolopendria extract, a plant used to treat Ovalbumin-specific IgE production was not significantly altered after respiratory disorders, is mainly caused by coumarins. Our results support treatment with umbelliferone, suggesting that this compound did not theprotectiveroleofcoumarinsinasthma. affect the production of IgE by activated B cells, in contrast to Studies on the pharmacokinetics of coumarin in man have shown dexamethasone, which caused a decrease on the levels of IgE. This there is extensive first-pass metabolism after oral administration effect of dexamethasone on IgE levels in the mouse model of allergic (Ritschel et al., 1979; Ritschel and Hoffmann, 1981; Ritschel, 1984). airway inflammation has been demonstrated before (Zhao et al., 2007). This, and other pharmacokinetic data in man, has led to suggestions In allergic asthma, the inhibition of macrophages, T lymphocytes, that coumarin is a prodrug, most likely active as umbelliferone or, eosinophils, mast cells, and mucus secretion leads to an improvement of possibly, even as the 7-hydroxyglucuronide form after metabolization respiratory flow. Altogether, our results show that umbelliferone in the liver (Hoult and Payá, 1996). Baccard et al. (2000) also attenuates airway inflammation in a murine model of asthma. The suggested that umbelliferone was vasodilating activity. Our results effects described herein, as well as those observed by other investiga- demonstrate that the effects of umbelliferone go beyond a broncho- tors, together with the broad spectrum of the biological effects of this dilating effect, since it has also potent immunomodulatory effects, substance, strongly suggest the therapeutic potential of umbelliferone downregulating cytokine and mucus production and inflammation. for the treatment of asthma and other allergic diseases. J.F. Vasconcelos et al. / European Journal of Pharmacology 609 (2009) 126–131 131

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